Kidney cross-sections of the cortex and medulla were dehydrated with isopropyl alcohol and infiltrated with paraffin in a tissue processor (Excelsior AS, Thermo Scientific, Inc.). Tissues were allowed to solidify in a base mold and then snapped into plastic tissue cassettes using a paraffin molding processor (HistoStar, Thermo Scientific, Inc.). Samples were cut to 5 μm thickness sections with a microtome (HM 355 S, Thermo Scientific, Inc.), and continuous “ribbon” of the sections were formed. Sections were carefully transferred onto a 40°C water dish for about 3–5 min to flatten and avoid wrinkles in the sectioned tissues.

Tissue slices were placed on the charged microscope slides, and deparaffinization was performed by placing the slides in a 60°C oven for 30 min in order to melt any extra paraffin. Slides were rinsed twice with a xylene solution for 5 min each, followed by hydration through a series of washing for 1 min each in decreasing alcohol concentrations (100, 95, 80, 70%). Slides were then submerged in the distilled water for 3 min for the staining process.

For H&E staining, tissues were stained with hematoxylin for 5 min, rinsed with tap water for 1 min and dipped for 1 sec in acid alcohol (200 ml of 50% alcohol containing 500 μL HCl 5N). Tissues were then stained with Eosin for 2 min and rinsed with distilled water for 1 min followed by dehydration with 95% and 100% alcohol each for 1 min and cleaning with xylene for 2 min. Coverslip was mounted onto a slide with xylene compatible mounting media.

For periodic acid Schiffs (PAS) staining, staining kit (Polysciences, Inc.) was used and experiments were performed based on the Company's protocol. Briefly, tissues were oxidized in 0.5% periodic acid for 5 min. After washing with deionized (DI) water, tissue sections were placed in Schiff's reagent for 15 min then rinsed with 0.55% potassium metabisulfite. Slides were washed in tap water for 10 min to allow the color to develop in the tissues, which were then counterstained with acidified Harris Hematoxylin for 30 s. Slides were washed with tap water until the tissues turned to a blue color. After dehydration with 95 and 100% alcohol each for 1 min and clearing with xylene for 2 min, the coverslips were mounted onto the slides with a xylene compatible mounting media.

For Masson's Trichrome staining, a staining kit (Polysciences, Inc.) was used according to the manufacture's instructions. Briefly, tissues were incubated in Bouin's fixative solution at 60°C for 1 h and rinsed with warm tap water for 2–3 min until the yellow color disappeared. Fresh Weigert's iron hematoxylin working solution was prepared by mixing Weigert's hematoxylin A and Weigert's hematoxylin B in a 1:1 ratio. Tissues were incubated in the working solution for 20 min and rinsed for 5 min with distilled water. Tissues were then stained with Biebrich Scarlet-Acid Fuchsin solution for 15 min and rinsed with tap water to remove any extra solution-residue. After rinsing with distilled water, the tissues were soaked in Phosphotungstic / Phosphomolibdic acid for 10 min, transferred to Aniline Blue for 7 min and washed with distilled water until the slides were clear. Tissues were then soaked in 1% acetic acid for 1 min and washed with distilled water for 1 min. After dehydration with 95 and 100% alcohol each for 1 min followed by cleaning with xylene for 2 min, the coverslips were mounted onto the slides with a xylene compatible mounting media.

For immunofluorescence staining, heat-induced epitope retrieval was performed using a pressure cooker and Tris-EDTA buffer (10 mM Tris base, 1 mM EDTA solution, 0.05% Tween 20, pH 9.0). Tris-EDTA buffer was first boiled in the pressure cooker to 100°C. Tissues were then maintained in the pressure cooker at about 121°C and 15 psi for 8 min. The pressure cooker and slides were then chilled quickly with cold tap water. The slides were rinsed three times with phosphate buffer saline (PBS) and blocked in PBS solution containing 5% bovine serum albumin for 15 min in a humidifying chamber. Using our standard protocol (Loghman-Adham et al., 2003; Nauli et al., 2003, 2006), tissues were stained with the ciliary marker acetylated-α-tubulin (1:1,000 dilution; Sigma-Aldrich, Inc.), distal tubular marker bolichos biflorus agglutinin (DBA; 4:1,000 dilution; Vector Laboratory, Inc.), proximal tubular marker lotus tetragonolobusl lectin (LTL; 4:1,000 dilution; Vector Laboratory, Inc.), and nucleus marker DAPI (Vector Laboratory, Inc.).

Isolation of renal epithelia from fresh tissues has been previously described in detail (Loghman-Adham et al., 2003; Nauli et al., 2003, 2006). After extensive washing in phosphate-buffered sucrose solution, the cortex and medullary regions of kidney were dissected into pieces and incubated with collagenase followed by 0.05% trypsin/0.53 mM EDTA at 37°C for 15–20 min. After vortexing vigorously for 5 min, ice-cold HBSS containing 10% FBS was added to inactivate the trypsin. The cells were further released from the fibrous basement membrane by trituration, washed twice with HBSS. Cells were then sorted based on their surface markers for DBA or LTL and resuspended in fresh culture medium. To allow attachment, the cells was grown in DMEM containing 10% FBS and supplemented with 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium, 36 ng/ml (10⁻⁷ M) hydrocortisone, 10⁻⁸ M triiodothyronine, 10 ng/ml EGF, and 50 ng/ml PGE1, as well as 100 U/ml penicillin, and 100 μg/ml streptomycin in a 37°C humidified incubator ventilated with 5% CO₂—95% O₂. The culture medium was changed every 2–3 days until confluency was reached.

Bending primary cilia with fluid-flow activates the cilium and increases cytoplasmic calcium in renal epithelial cells (Praetorius and Spring, 2001; Liu et al., 2005; Jin et al., 2014). Thus, intracellular calcium was measured with Fura2-AM [to study cilia function] as previously described (Nauli et al., 2013). Briefly, after pre-incubation with 5 μM Fura2-AM for 30 min at 37°C, the tissues were equilibrated for at least a minute. The optimal shear-stress of 0.8 dyne/cm² was used to monitor changes in cytosolic calcium as previously described (Nauli et al., 2013). Ionomycin (1 μM) was used at the end of each experiment to confirm Fura-2 loading and determine minimal and maximal ratiometric fluorescence signals. Cells were then analyzed for cilia length by staining with the ciliary marker acetylated-α-tubulin by staining with the ciliary marker acetylated-α-tubulin as previously described (Loghman-Adham et al., 2003; Nauli et al., 2003, 2006).

All images represent the extracted information from the digital pictures. Images were captured through a Nikon Ti-E microscope. The Nikon NIS Elements for Advanced Research software was used for image capture and analysis, including automatic object recognition, image scanning and color binary segmentation. Scale bars were provided in all figures to indicate the actual image reduction size. All morphometric data were reported as mean ± SEM. Distribution analyses were performed and presented on all data sets to verify normal data distributions. After distribution and variance analyses, data comparisons for more than two groups were performed using ANOVA test followed by a Tukey post-hoc analysis. The correlation between cilia length and cilia function was analyzed with the ordinary least squares (OLS) regression of y on x, because the ordinary least products (OLP) would not allow analysis of covariance as a technique for testing the equality of slopes or intercepts in linear regressions (Ludbrook, 2012). Pearson correlation coefficients were therefore used to analyze the significance of differences in the coefficients correlating primary cilia length and function. Asterisks (^*) denote with statistical significance differences at p < 0.05 relative to corresponding control groups as indicated in the figures.

A total of 12 sheep and 12 lambs was used; for each normoxic or hypoxic group, 6 sheep and 6 lambs were used (N = 6 sheep and N = 6 lambs for normoxia; N = 6 sheep and N = 6 lambs for hypoxia). From each animal, both kidneys were collected for different studies. Each pair of kidneys were randomly selected for an immediate fixation in formalin or trypsinization for cell culture. From each kidney, a minimum of 10 experimental replicates was sampled. All statistical analysis was done with GraphPad Prism, version 5.0b.

Representative images of H&E stained renal tissue sections from normoxic and hypoxic kidneys are presented (Figure 1A). In all kidney sections, light microscopy analysis indicated that the cortex regions of the kidneys had normal glomeruli. There were no significant differences in glomerulus size between normoxic and hypoxic kidneys (Figure 1B). Capillary loops of the glomeruli were also normal, and the number and morphology of endothelial cells were comparable between normoxic and hypoxic tissues.

In normoxic fetal kidneys, all tubules were closely spaced in the interstitium. Tubules were lined with a single layer of epithelia and well-organized nuclei. In hypoxic fetuses, the renal cortex demonstrated some evidence of mesangial and intercapillary cell proliferation. The renal medulla showed apparent atrophic tubules, interstitial fibrosis and edema, and tubular disparition characterized by greater inter-tubular spaces and separation of tubules filled with edema (Figure 1C).

While all tubules in adult sheep were closely spaced in the interstitium and lined by a single layer of cells with well-organized nuclei, some abnormalities were observed in the chronically hypoxic kidney. The hypoxic kidneys showed some proliferation of mesangial cells in the cortex and slight tubular edema in the medullary region. Otherwise, the surrounding tubules appeared normal in both normoxic and hypoxic adult kidneys.

PAS staining was done to highlight basement membranes of glomerular capillary loops and tubular epithelia. Renal tissue sections from normoxic and hypoxic kidneys are shown in the representative images (Figure 2A). There were no significant differences in basement membranes thicknesses of glomerular capillary loops between normoxic and hypoxic adult kidneys.

Basement membrane thicknesses of the tubules were significantly less in hypoxic fetal kidneys compared to normoxic fetal kidneys (Figure 2B). Although a reverse trend was observed in adult kidneys, it was not statistically significant in adult normoxic or hypoxic kidneys. To refine the statistical analysis, tubular membrane thicknesses were re-measured by discriminating the tubular origins. In proximal tubules, no significant difference in tubular membrane thickness was observed (Figure 2C). In distal collecting tubules, tubular membranes were significantly thicker in hypoxic than normoxic adult kidneys. As expected, tubular membrane thicknesses were significantly less in hypoxic compared to normoxic fetal kidneys.

As observed in the H&E staining, hypoxic fetal kidneys showed interstitial medullary edema, fibrosis, tubular disparition and atrophy characterized by greater inter-tubular space (Figure 2D).

To examine potential fibrosis in the kidneys, Masson's Trichrome staining was performed to highlight deposition of collagen and fibrin fibers. Renal tissue sections from normoxic and hypoxic kidneys are shown in representative images (Figure 3A). The percentage of blue-stained area was calculated using a binary threshold program, which indicated significant fibrosis in hypoxic compared to normoxic kidney tissues (Figure 3B). In normoxic tissues, there was some collagen fibers that were normally distributed around renal tubules. In hypoxic tissues, normal distributions of collagen fibers were observed, but abnormal depositions of collagen/fibrin fibers were also observed throughout tubular epithelia and interstitial spaces.

As observed in the H&E and PAS staining, hypoxic fetal kidney showed interstitial medullary edema characterized by more inter-tubular space (Figure 3C). As also seen in previous staining, hypoxia modulated lumen size of distal tubules in both fetal and adult kidneys (Figure 3D). No significant changes in lumen size were observed in proximal tubules.

It remains uncertain if hypoxia can maintain and stabilize the primary cilia or it would inhibit primary cilia formation (Verghese et al., 2011; Ding et al., 2015; Lavagnino et al., 2016; Resnick, 2016). To investigate the effect of chronic hypoxia, primary cilia were labeled with the cilia marker acetylated-α-tubulin. Representative images of renal tissue sections from normoxic and hypoxic kidneys are shown for staining of cilia and tubular markers. Distal collecting (Figure 4A) and proximal (Figure 4B) tubular markers were used to identify cilia length in respective tubules. Cilia measurements were the represented in the bar graphs to compare the distributions of the cilia length (Figure 4C). While hypoxia did not significantly alter cilia length in adult kidneys, fetal kidneys were very susceptible to hypoxia (Figure 4D). Cilia length was significantly longer in hypoxic fetal kidneys than normoxic fetal kidneys.

Of note, that the distal collecting tubules had larger lumen sizes in adult than fetal kidneys. This may be associated with longer cilia in adult than fetal distal collecting tubules. For the first time, our studies show that the length of primary cilia in distal collecting tubules become longer during maturation, while there is no change in proximal tubule cilia length from fetuses to adults.

Renal primary cilia are mechanosensory organelles that sense filtrate moving within the tubules. To examine the possibility that hypoxia could alter mechanosensory of cellular responses, renal epithelia were isolated, cultured, stained with a cilia marker and challenged with shear-stress. Representative images of these cells from normoxic and hypoxic kidneys reveal changes in cilia length (Figure 5A). On average about 80% of cells had cilia, and there were no apparent differences in cilia formation between normoxic and hypoxic tissues, or between fetal and adult kidneys. Cilia measurements were then tabulated to compare their distributions (Figure 5B). Cilia measurements were also tabulated to analyze the impact of hypoxia on cilia lengths (Figure 5C). While hypoxia did not significantly alter cilia length in adult cells, cilia length was significantly longer in hypoxic than normoxic fetal cells. Interestingly, trend in cilia length changes was similar for in vitro and in vivo preparations. When the overall cilia length in vivo kidney (Figure 4) or in vitro cell culture (Figure 5) were averaged, it was apparent that the in vitro cell culture produced much longer cilia than observed in vivo (6.35 ± 0.28 μm vs. 2.84 ± 0.12 μm; p < 0.00005).

To examine the effect of chronic hypoxia on mechanosensory cilia function, cells were challenged with 0.8 dyne/cm² shear-stress. Changes in cytosolic calcium were averaged and plotted in line graphs (Figure 6A). When peaks of cytosolic calcium were examined, hypoxia significantly enhanced mechanosensory function in fetal epithelial cells (Figure 6B). In contrast, hypoxia did not significantly alter mechanosensory sensitivity in adult cells.

It has been hypothesized that longer cilia are more sensitive to fluid-shear stress (Abdul-Majeed et al., 2012; Upadhyay et al., 2014). To examine the effect of hypoxia on cilia length-function relationship, both in vivo (Figure 4) and in vitro (Figure 5) cilia lengths were used to assess changes in cilia function. When in vivo cilia length was plotted against cilia function, no apparent length-function association was observed (Figure 7A; R² = 0.42). Because hypoxia did not alter cilia length in adult kidneys, length-function relationship was next analyzed only in fetal kidneys. This revealed significant correlation within length-function relationships (Figure 7B; R² = 0.86). When in vitro cilia length was analyzed, the cilia function was significantly correlated with cilia length following either inclusion (Figure 7C; R² = 0.79) or exclusion (Figure 7D; R² = 0.93) of adult kidneys.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.